EP1987347A1 - Quadrature en ligne et detection interferrometrique en quadrature de phase amelioree antireflet - Google Patents

Quadrature en ligne et detection interferrometrique en quadrature de phase amelioree antireflet

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Publication number
EP1987347A1
EP1987347A1 EP07757051A EP07757051A EP1987347A1 EP 1987347 A1 EP1987347 A1 EP 1987347A1 EP 07757051 A EP07757051 A EP 07757051A EP 07757051 A EP07757051 A EP 07757051A EP 1987347 A1 EP1987347 A1 EP 1987347A1
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Prior art keywords
probe beam
substrate
support layer
spots
beam waves
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English (en)
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David D. Nolte
Manoj Varma
Ming Zhao
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Purdue Research Foundation
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Purdue Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N2021/4704—Angular selective
    • G01N2021/4707—Forward scatter; Low angle scatter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7773—Reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7779—Measurement method of reaction-produced change in sensor interferometric

Definitions

  • the present invention generally relates to apparatus, methods and systems for detecting the presence of one or more target analytes or specific biological material in a sample, and more particularly to a laser scanning system for detecting the presence of biological materials and/or analyte molecules bound to target receptors on a disc by sensing changes in the optical characteristics of a probe beam reflected from the disc by the materials and/or analytes.
  • immunological compact disk which simply includes an antibody microarray.
  • Ekins, R., F. CIm, and E. Biggart Development ofmicrospot multi-analyte ratiometric immunoassay using dual ⁇ oures cent- labelled antibodies.
  • Ekins, R. and F.W. Chu Multianalyte micro spot immunoassay - Microanalytical "compact Disk” of the future. Clin. Chem., 1991 , Vol. 37(11), p.
  • Interferometric optical biosensors have the intrinsic advantage of interferometric sensitivity, but are often characterized by large surface areas per element, long interaction lengths, or complicated resonance structures. They also can be susceptible to phase drift from thermal and mechanical effects.
  • One embodiment according to the present invention includes an apparatus for use with an optical probe beam and a detector for detecting the presence of a target analyte in a sample.
  • the apparatus includes a substrate and a biolayer located on the substrate, the biolayer consisting of a distribution of molecular dipoles; or alternatively having an effective thickness and a refractive index; and the substrate having a reflection coefficient.
  • the magnitude of the substrate reflection coefficient is substantially minimized.
  • the substrate can include a dielectric material including silicon or a silicon dioxide layer on silicon.
  • the biolayer and the substrate can be designed such that the scattered wave from the probe beam hitting the target analyte is substantially in-quadrature with the reflected wave from the probe beam hitting the substrate.
  • the biolayer and the substrate can be designed to substantially maximize the electric field strength at the surface of the biolayer
  • Another embodiment according to the present invention includes an apparatus for use with a probe beam and a detector for detecting the presence of a target analyte in a sample, where the apparatus includes a biolayer; and a structure comprising a support layer on a substrate.
  • the magnitude of the substrate reflection coefficient is substantially minimized.
  • the thickness of the support layer can be selected such that the scattered wave from the top of the support layer is substantially out-of- phase with the reflected wave from the bottom of the support layer.
  • a further embodiment according to the present invention includes a method for detecting the presence of a target analyte in a sample.
  • the method includes providing a substrate having a plurality of analyzer molecules distributed about the substrate; contacting a sample to at least some of the analyzer molecules; scanning the substrate with a probe beam; and detecting one of the presence or absence of a target analyte in the sample based on the reflected signal from the probe beam; wherein the detecting includes conversion of phase modulation into intensity modulation at the detector.
  • a further embodiment according to the present invention includes an apparatus for use with an optical probe beam and a detector for detecting the presence of a target analyte in a sample.
  • the apparatus includes a substrate and a biolayer located on the substrate, the biolayer having a refractive index and the substrate having a reflection coefficient.
  • the biolayer and the substrate can be designed such that the scattered wave from the probe beam hitting the target analyte molecules is substantially in-phase with the reflected wave from the probe beam hitting the substrate surface.
  • a further embodiment according to the present invention includes an apparatus for use with a probe beam and a detector for detecting the presence of a target analyte in a sample, where the apparatus includes a biolayer; and a structure comprising a support layer on a substrate.
  • the thickness of the support layer can be selected such that the scattered wave from the top of the support layer is substantially in quadrature with the reflected wave from the bottom of the support layer.
  • the method includes detecting one of the presence or absence of a target analyte in the sample based on the reflected signal from the probe beam; wherein the detecting includes direct conversion of phase modulation into intensity modulation.
  • the detecting can be done without apertures or split detectors.
  • the detecting can include detecting the scattered wave returned from the target analyte and the reflected wave returned from the substrate, the scattered wave being substantially in-phase with the reflected wave.
  • a further embodiment according to the present invention includes an apparatus for use with a probe beam and a detector for detecting the presence of a target analyte in a sample, where the apparatus includes a biolayer; and a structure comprising a support layer on a substrate.
  • the thickness of the support layer can be varied across the substrate such that the phase relationship between the waves reflected from the top and the bottom of the support layer can vary continuously between the condition of phase quadrature and the condition of being in-phase.
  • the detecting can be done both with and without apertures or split detectors to convert the phase modulation caused by the target analyte into intensity modulation at the detector.
  • FIG. 1 is a schematic illustration of reflection and scattering caused by a molecule on a mirror and the combination of scattered and reflected waves in the far field;
  • FIG. 2 is an illustration of wavefunctions and boundary conditions of a uniform layer of molecules on a mirror
  • Fig. 3 is a graph illustrating differential phase-contrast intensity modulation for a monolayer biofilm on an antireflection structure as a function of the support thickness for different substrate refractive indexes;
  • Fig. 4 is a graph illustrating reflectance versus support layer thickness for the conditions in Figure 3;
  • Fig. 5 is a graph illustrating absolution intensity modulation versus support layer thickness, the product of Figure 3 with Figure 4;
  • Fig. 6 A is a graph illustrating squared electrical field versus position for gold on glass
  • Fig. 6B is a graph illustrating relative intensity modulation for gold on glass versus gold thickness in response to a monolayer
  • Fig. 7 is a graph illustrating phase modulation and reflectance modulation versus top layer thickness caused by a bio monolayer on a dielectric stack
  • Fig. 8 is a graph illustrating phase and reflectance versus thickness for an anti- reflection layer on ZrOo with and without a biolayer
  • Fig. 9 is a graph illustrating relative intensity modulation versus support thickness for antireflection layer on ZrO 2 ;
  • Fig. 10 is a graph illustrating electrical field strength at the surface of silicon versus position for real, imaginary, and total magnitude electric filed components
  • Fig. 11 is a graph illustrating squared electrical field strength versus position for a silicon surface and an anti-node surface
  • Fig. 12 is a graph illustrating electrical field strength versus position at the surface of quarter-wave oxide-on-silicon with and without an antibody monolayer;
  • Fig. 13 is a graph illustrating phase shift caused by the biolayer and reflectance as a function of oxide thickness on silicon;
  • Fig. 14 is a graph illustrating differential phase contrast and direct intensity modulation in response to an 8 nm monolayer of antibody versus oxide thickness showing the response of the phase and intensity channels and their summation in quadrature;
  • Fig. 15 is a graph illustrating electric field versus position for an anti-reflection- coated silicon surface with and without an antibody layer;
  • Fig. 16 is a graph illustrating differential phase contrast and direct intensity modulation caused by an antibody biolayer
  • Fig. 17 is a schematic drawing of the disc structure of an embodiment of the inline biological disc and the reflection of light rays therefrom;
  • Fig. 18 is a graph illustrating the intensity shift caused by 1 nm of protein versus oxide thickness for several different wavelengths
  • Fig. 19A is a graph illustrating the intensity shift produced by protein measured directly as a time trace of total light intensity;
  • Fig. 19B shows a two dimensional surface profile obtained by putting time traces taken at consecutive radii together into a 2D display;
  • Fig. 2OA is a graph illustrating a distribution of assay signal for each unit cell as a function of dose for an embodiment of the in-line system
  • Fig. 2OB is a graph illustrating a the dose response curve for an embodiment of the in-line system
  • Fig. 21 is a graph illustrating the measurement error versus the number of assays per disc
  • Fig. 22 is a graph illustrating the concentration detection limit set by the measurement error and the response curve
  • Fig. 23 shows a cross section across a single spot showing an outer ridge and internal ridges
  • Fig. 24 shows a high-resolution scan of a spot with a clear ring structure
  • Fig. 25 is a schematic illustration and a graph of improved discrimination between molecular phase and Rayleigh scattering at 120 nm oxide thickness
  • Fig. 26 shows a spatial scan of approximately 200 spots on a 120 nm oxide biological disc across 2.5 mm with spot diameters of approximately 120 microns and heights of about 3 nm;
  • Fig. 27 shows an example of a "unit cell” with target and reference spots placed in a 2x2 array, and the data on the right shows unit cell spots of approximately 120 micron diameter printed onto a 120 nm oxide biological disc;
  • Fig. 28 shows an image subtraction protocol with a postscan image being subtracted from a prescan image to produce a resultant difference image on the right showing the change in surface height
  • Fig. 29 shows an the detection sensitivity of in-line quadrature on a 120 nm oxide biological disc, the scan data on the upper left providing two line plots on the right, one through the center of an IgG spot, and the other on the so-called land;
  • Fig. 30 shows a histogram of the root height variance between two scans of the same disc before and after a 20 hour buffer wash
  • Fig. 31 shows an embodiment of a disc layout with 25,600 spots placed in a 2x2 unit cell pattern with 100 radial spots and 256 angular spokes;
  • Fig. 32 is assay data showing change in spot mass as a function of analyte concentration for a series of incubations on a 120 nm oxide disc, the curve being a fit to a
  • 11/345,462 entitled “Method and Apparatus for Phase Contrast Quadrature Interferometric Detection of an Immunoassay,” filed February 1, 2006; and also U.S. Patent Application Serial No. 11/345,477 entitled “Multiplexed Biological Analyzer Planar Array Apparatus and Methods,” filed February 1, 2006; and also U.S. Patent Application Serial No. 11/345,564, entitled “Laser Scanning Interferometric Surface Metrology,” filed February 1, 2006; and also U.S. Patent Application Serial No. 11/345,566, entitled “Differentially Encoded Biological Analyzer Planar Array Apparatus and Methods,” filed February 1, 2006, the disclosures of which are all incorporated herein by this reference.
  • quadrature might be narrowly construed as what occurs in an interferometric system when a common optical "mode” is split into at least 2 "scattered” modes that differ in phase by about N* ⁇ /2 (N being an odd integer).
  • an interferometric system is in quadrature when at least one mode "interacts" with a target molecule and at least one of the other modes does not, where these modes differ in phase by about N* ⁇ /2 (N being an odd integer).
  • This definition of quadrature is also applicable to interferometric systems in which the "other mode(s).” referring to other reference waves or beams, interact with a different molecule.
  • the interferometric system may be considered to be substantially in the quadrature condition if the phase difference is ⁇ /2 (or N* ⁇ /2, wherein N is an odd integer) plus or minus approximately twenty or thirty percent.
  • Summing in quadrature is a separate use of the term "quadrature" not directly related to phase quadrature of interferometry. Two independent signals are summed in quadrature by taking the sum of their squared magnitudes. Summing in quadrature is a method for taking two varying output signals that arise from varying properties of a system being measured, and combining them into a single measurement that is substiantially constant.
  • in-phase in the present invention is intended to describe in-phase constructive interference, and "out of phase” is intended to describe 180-degree-out-of-phase destructive interference. This is to distinguish these conditions, for both of which the field amplitudes add directly, from the condition of being "in phase quadrature” that describes a relative phase of an odd number of ⁇ /2.
  • Optical interferometric detection of biomolecules at surfaces depends on the phase shift imposed by the molecules on a probe optical field. For a monolayer of macromolecules such as antibodies on a typical surface such as glass this phase shift is typically only a few percent of a radian. This small phase shift produces a detected intensity modulation of only a few percent when operating in interferometric quadrature. Treatment of surfaces with dielectric layers can enhance the molecular phase shift and the relative intensity modulation in quadrature interferometry. Immobilization of molecules on anti-nodal high -reflectivity mirrors produces enhancements of about three times. Immobilization of molecules on anti- reflection surfaces, on the other hand, can produce an enhancement of about fifteen times.
  • the scattering coefficient f is real and in phase with the exciting field E 0 .
  • the total far field, including contributions from both the direct wave and the scattered wave, is given by:
  • phase shift associated with scattering from a single molecule This is the phase shift associated with scattering from a single molecule.
  • the phase can be attributed to a refractive index of the molecular medium.
  • the medium becomes more dense, local-field corrections modify the molecular scattering through depolarization fields, but the basic origin of refractive index is in the molecular scattering.
  • Interfeiometric optical biosensors can be used to detect the phase shift on a probe field caused by the presence of biomolecules.
  • ⁇ 635 nm and the refractive index of the biolayer n ⁇ 1.3
  • the phase shift caused by molecular scattering at surfaces can be enhanced by reducing the contribution of the direct field, while keeping the molecularly scattered field constant.
  • phase modulation into intensity modulation at the detector is the combination of the probe wave (carrying the phase modulation from the biolayer) with a reference wave that is in phase quadrature (or 90° relative phase). In the condition of quadrature, the intensity modulation at the detector is a maximum and depends linearly on the amount of phase modulation.
  • One method to attain the quadrature condition is to detect phase modulation through the observation of two waves, one passing through the analyte and one falling on the substrate adjacent to the analyte, at an angle called the quadrature angle.
  • the two waves at the quadrature angle are in quadrature, and the intensity change is directly proportional to the protein height. This is called phase-contrast quadrature and acquires a differential phase contrast signal.
  • the anti-reflection enhancement of molecular phase shift described in the preceding paragraphs represents a new embodiment of differential phase contrast quadrature.
  • the differential phase signal is enhanced by reducing the reflectance of the supporting substrate.
  • a second method to attain the quadrature condition is to detect the phase modulation directly by designing the substrate to have a reflection coefficient that is shifted in phase by 90 degrees. This condition is in-between the nodal and anti-nodal conditions.
  • the reflected field has a 90 degree phase shift in the near field, the reflected reference and the scattered molecular signal become in phase in the far field, interfering and directly creating intensity modulation.
  • no differential phase contrast scheme is needed to detect it. Surface analytes can be measured directly.
  • This form of direct quadrature detection is closely related to the case of anti- reflection coatings.
  • the support layer is a little off the quarter-wave condition corresponding to a reflectance minimum, the reflected wave can have the required 90 degree phase shift, creating the condition for direct detection in the far field without the need for quadrant detectors. Therefore, by operating near a reflectance minimum condition, the differential phase contrast and this direct detection of phase both benefit from the anti- reflection enhancement.
  • Anti-reflection enhancement of differential phase contrast describes the enhanced detection of differential phase contrast signals caused by placing the molecules or biolayers on a substrate substantially in or near an anti-reflectance condition.
  • In-line quadrature describes the direct phase-to-intensity conversion that occurs when the wave scattered from the target analyte molecules are substantially in-phase with the wave reflected from the substrate.
  • the signal-to- noise ratio in addition to the phase shift, also impacts interferometric detection. This depends on the specific noise contributions such as relative intensity noise (RTN), shot noise and system noise.
  • RTN relative intensity noise
  • the signal-to-noise increases as r goes to zero. Therefore, the decreasing photon flux does not impact the increased sensitivity, and the best condition in this case is an anti-reflection surface. Low reflectance can be offset by higher laser power.
  • the signal-to-noise ratio is: which goes to zero as r goes to zero. This is therefore not advantageous, and the best condition in this case is high reflectance with an anti-node surface and using differential phase contrast detection.
  • the signal-to-noise ratio is:
  • (SN) is a coefficient related to the shot noise magnitude. This S/N is independent of r in the small-r limit and is comparable to the free-space case of molecular phase shift. [0071] Therefore, from the point of view of signal-to-noise performance, if the system noise can be reduced so that relative intensity noise dominates, then the anti-reflection condition gives the best enhancements in S/N. Low photon flux can be compensated by higher power laser sources and by lower-intensity detectors such as APDs. Anti-reflection coatings can also be more economical than multi-layer mirror stacks.
  • the molecular layer becomes dense, it may more appropriately be modeled by a thin homogeneous layer with a refractive index n.
  • Figure 2 will be used to discuss a biolayer on a substrate with reflection coefficient ro in the absence of the layer that can be a complex value.
  • the uniform layer has a thickness "d" and refractive index n p .
  • the fields in the incident half-space and the protein layer are:
  • This formula can be used to calculate the relationship between the reflection coefficient r between the protein layer and the substrate and the "bare" reflection coefficient r 0 of the substrate as:
  • the additive term is the phase modulation of the layer that is also the molecularly scattered wave. This shows that, when the second term is in phase with r 0 , the condition of in-line quadrature holds. And, when the second term is in quadrature with ro, the condition of differential phase contrast holds.
  • the protein profile is either the odd derivatives, for differential phase contrast, or the even derivatives, for in-line quadrature. Both cases benefit from small reflectance because of the r 0 term in the denominator, and hence both are enhanced by working at or near a reflectance minimum.
  • the phase of the wave scattered from the target analyte molecules is related to the phase of the wave scattered from the substrate.
  • in-phase quadrature results.
  • in quadrature results.
  • differential phase contrast results.
  • the difference between these two conditions is set by the phase of ro-
  • the substrate is composed of a support layer on a base material.
  • the molecules or biolayers are on top of the support layer.
  • the refractive index of the support layer can be chosen to substantially minimize the reflectance (the magnitude of the reflection coefficient).
  • the thickness of the support layer can be varied to tune the phase of the reflection to bring the detection into in-line quadrature or into differential phase contrast.
  • the simplest anti-reflection surface is the single quarter wave layer on a substrate with a reflection coefficient of: _ Ti 1 (n 0 — n s ) cos kh + i(n o n s - tif ) sin kh H 1 (Ii 0 + n s )coskh + i(n Q n s +nf)siakh for n s the refractive index of the base, ni the index of the support layer, and no the index of the top space.
  • the reflection coefficient goes to zero at the anti-reflection condition for a quarter-wave layer under the condition:
  • phase of the simple anti-reflection surface is real (in phase quadrature with the waves scattered from the target analyte molecules) when the support layer has a quarter- wave thickness. This gives the anti-reflection enhancement of differential phase contrast.
  • the support layer has a thickness of approximately an eighth of a wavelength, then the phase of the reflection coefficient becomes a purely imaginary number and the reflected wave is in-phase with the wave scattered by the molecules. Tn this case one sees that the wave reflected from the top of the support layer and the wave reflected from the bottom of the support layer are in phase quadrature. This is the condition of in-line quadrature.
  • in-line quadrature has two modes of description that are mutually self- consistent.
  • the inline quadrature condition When viewed as a molecule on a substrate with a reflection coefficient, the inline quadrature condition is attained when the wave scattered from the molecule and the wave reflected from the substrate are in phase. When viewed as a molecule on a support layer, the in-line quadrature condition is attained with the wave reflected from the top of the support layer and the wave reflected from the bottom of the support layer are in phase quadrature.
  • FIG. 3 shows the anti-reflection-enhanced differential phase-contrast intensity modulation for a monolayer biofilm on an antireflection structure as a function of the support thickness.
  • the support is index-matched to the biolayer, and the substrate refractive indexes are shown.
  • Figure 4 shows the corresponding reflectances for the conditions shown in Figure 3. As the substrate index approaches 1.82, the reflectance goes to zero. The low reflectance at the anti-reflection condition reduces the absolution intensity modulation, which is shown in Figure 5.
  • the absolute intensity modulation is the product of intensity modulation in Figure 3 and the reflectance in Figure 4.
  • the absolute signal decreases as the anti-reflection condition is approached. As long as the relative intensity noise of the laser continues to dominate, the S/N is not adversely affected by the decreased absolution photon flux. Silicon as the substrate provides a balance between enhancing and decreasing absolute signals.
  • the most general situation involving multiple layers in the substrate and in the biolayer is modeled using the transfer matrix approach. Realistic complex refractive index of actual materials are easily incorporated in this approach. Common materials and substrate structures include gold, quarter- wave dielectric stacks, anti-reflection surfaces, and silicon with thin or thick oxides or other coatings.
  • Thick gold behaves very close to a nodal high-reflectance surface.
  • the presence of the field null near the surface makes biolayers nearly "invisible" on this surface.
  • the squared field is shown in Figure 6A for gold on glass with a gold thickness of 80 nm.
  • the intensity at the surface of the gold is 0.5 compared with 4 for a perfect anti-nodal mirror, and decays rapidly inside the gold with a decay length of 16 nm.
  • the relative intensity modulations for both differential phase contrast and in-line quadrature for gold on glass as a function of gold thickness are shown in Figure 6B.
  • the differential phase contribution to the intensity modulation is shown in Figure 6B to be only 3% for a thickness of 16 nm.
  • Figure 6B also shows that at a thickness of about 3 nm of gold on glass, there is nearly a 30% differential phase contrast signal from a bio monolayer.
  • the in-line intensity modulation is almost 20% for thicknesses slightly large and smaller than 3 nm.
  • gold of this thickness tends to aggregate rather than being a uniform layer.
  • Gold on silicon, instead of glass, on the other hand does not lead to high phase shifts because of the large refractive index of silicon.
  • Dielectric quarter wave stacks are readily designed to have high reflectance, as well as control over the reflected phase.
  • the two most common phase conditions are nodal- surfaces and anti-nodal- surfaces. In between these two conditions comes the case when the reflection coefficient takes on purely imaginary values and hence are in the in-line quadrature condition.
  • Figure 7 shows phase modulation and reflectance modulation caused by a bio monolayer on a dielectric stack. The surface begins as an anti-nodal condition, and goes to a nodal condition. In between is the "in-line" condition for which the reflectance modulation is negligible. This is because the high reflectance cannot be modified by the phase shift induced by the biolayer.
  • in-line quadrature does not apply to the case of high- reflectance substrates.
  • the anti-nodal surface gives a phase shift roughly twice the double-pass phase from the layer.
  • the enhanced differential phase contrast peak is also relatively broad with a FWHM of almost 100 nm, making the surface insensitive to slight drifts in layer thickness.
  • An anti-reflection surface can be obtained using quarter- wave layers on a substrate which can provide nearly perfect impedance matching to the substrate, driving reflectance to nearly zero.
  • This anti-reflection surface enhances the phase shift caused by a biolayer on a surface.
  • the phase and reflectance for this structure with and without a biolayer is shown in Figure 8.
  • the phase jump near the reflectance minimum is pronounced in this case, and the effect of the biolayer is large.
  • the relative intensity modulation for this structure is shown in Figure 9. There are two contributions: one from the differential phase contrast, and one directly from the in-line amplitude modulation from the surface.
  • the anti-reflection enhancement of the differential phase contrast is very large at the anti-reflection condition.
  • the in-line effect is also large for this structure because the scattered wave and the reflected wave can be in phase when slightly off the anti-reflection condition, adding constructively in the far field.
  • This in-line condition is direct quadrature which is not differential and hence gives absolute protein heights.
  • the FWHM width of the enhancement is about 25 nm.
  • the FWHM is broader for the total modulation, providing more stability for the detection method.
  • Silicon is one of the most common materials available because of its importance to the electronics industry. It therefore is a good substrate choice for economic reasons, as well as for its compatibility with anti-reflection coatings.
  • Figure 11 depicts the squared field strength at a silicon surface compared to an anti-node surface.
  • the squared field on silicon is 60% of the anti-node case.
  • the calculated phase shift for silicon is 8% of the anti-node case.
  • bare silicon is not a useful surface for interferometry.
  • thermally-grown silicon dioxide on silicon provides a strong refractive index difference between both air/oxide and oxide/silicon interfaces.
  • the oxide thickness is a quarter- wavelength ⁇ /4*N (N being an odd integer) in thickness
  • the electric field is a maximum at the oxide surface (anti-node) where the field is maximally sensitive to an added biolayer. This is illustrated in Figure 12 showing the electric field strength for a quarter-wave oxide on silicon with and without an antibody layer.
  • the surface is anti-nodal and hence has a field maximum and the condition of differential phase contrast.
  • the phase shift is 0.226 rad caused by the biolayer, which is about 20 times larger than for bare silicon (which has nearly a nodal surface).
  • the sensitivity of phase and reflectance as a function of the oxide thickness on silicon is shown in Figure 13.
  • the near-anti-reflection condition is at the quarter-wave thickness of 100 nm.
  • FIG. 14 The intensity modulation in response to an 8 nm monolayer of antibody is shown in Figure 14 as a function of the oxide thickness.
  • Figure 14 shows the phase channel (assuming quadrature detection of the phase modulation), the amplitude channel (detecting the full far-field intensity), and a quadrature sum of these two channels.
  • An interesting application of the summed quadratures occurs if the disk thickness is varied across the disk. The summed quadrature is less sensitive to thickness variations than either of the individual channels.
  • the differential phase contrast channel for a thick biolayer can have over 30% intensity modulation.
  • the combined channels have a broad bandwidth that provides stability against varying oxide thickness across the wafer.
  • Figure 15 shows the electric field for an anti-reflection-coated silicon surface.
  • the electric field for an antireflection condition is nearly unity (no reflection), but this condition is spoiled by the antibody layer that reflects light.
  • Figure 16 shows the differential intensity modulation caused by the antibody biolayer.
  • the differential intensity modulation can be arbitrarily large because the original reflectance can be arbitrarily close to zero. Phase wrapping occurs in this case, as shown in Figure 16, with multiple peaks as a function of oxide thickness. The intensity modulation can be over 100%.
  • the in-line intensity channel in Figure 14 shows the performance of the new quadrature class called In-line Quadrature.
  • the phase modulation caused by the biolayer is converted directly to intensity modulation.
  • the peaks of this in-line response occur at 80 nm and 120 nm.
  • the quadrature condition for in-line detection is at approximately an eighth-wave thickness, for ⁇ /8*N where N is an odd number and the wavelength is the wavelength in the support layer (free-space wavelength divided by the refractive index of the layer).
  • the field amplitude is maximum (anti-node) at a quarter wave, and decreases to zero at zero-wave or half- wave.
  • One embodiment of the in-line quadrature class uses a silicon wafer coated by a layer of SiO 2 as a substrate for immobilized biomolecules.
  • the thickness of the SiO 2 layer is chosen so that light reflected from the SiO 2 surface on top and light reflected from the silicon surface below is approximately in phase quadrature.
  • Protein molecules scatter the incident light, adding a phase shift linearly proportional to the mass density of the immobilized protein, which is converted to a far-field intensity shift by quadrature interference. Patterning of protein can be done by spot printing with a jet printer, which can produce protein spots 0.1 mm in diameter.
  • the typical 1/f system noise has a 40 dB per octave slope, and at a frequency well above the 1/f noise, a 50 dB noise floor suppression can be obtained, making it possible to measure protein signals with high precision.
  • Fig. 17 shows a schematic of light rays reflected from the disk structure of an embodiment of an in-line quadrature system. It is based on the quadrature interference of light reflected from the top oxide (SiO 2 ) surface and from the bottom silicon (Si) surface. The phase difference of these two beams is set by the oxide thickness. When the oxide thickness is approximately ⁇ /8 or 3 ⁇ /8, the two beams are in quadrature. The presence of protein scatters the incident beam and adds an optical phase shift, which is then converted to a far-field intensity shift. The intensity shift not only depends on the quadrature interference, but also on the surface electric field strength, and the actual protein signal is a combination of these two factors.
  • In-line quadrature disks can be fabricated from 100-mm diameter silicon wafers with a layer of thermal oxide.
  • the thickness of the SiO 2 layer is chosen to be 80 nm or 120 nm to obtain close to a ⁇ /2 or 3 ⁇ /2 phase quadrature condition when using a 635 nm wavelength divided by the refractive index of silica.
  • the 3 ⁇ /2 quadrature is preferred, because by working at this quadrature, the intensity shift caused by the presence of protein is positive, thus easily distinguishing it from scattering from dust or salt particles, which has negative signal.
  • the SiO 2 surface can be functionalized with an isocyanate coating which binds protein covalently.
  • the optical detection system uses a 635nm diode laser as the light source.
  • the laser beam is focused onto the disc by a 5 cm focal length objective to a 20 micxon diameter.
  • the disc is mounted on a stable spinner, such as one available from Lincoln Laser Inc. of Phoenix, AZ, and spun at 20 Hz.
  • the reflected light from the disc is collected by the same objective and directed to a photodetector by a beam sputter.
  • the detector is a quadrant detector that has three output channels: one total intensity channel and two difference channels (left minus right, and top minus bottom). For in-line operation, only the summed intensity channel is used for detection, while the other two channels provide diagnostics for optical alignment.
  • the intensity shift produced by protein is measured directly as a time trace of total light intensity, as shown in Figure 19 A.
  • a two dimensional surface profile can be obtained by putting time traces taken at consecutive radii together into a 2D display, as is shown in Figure 19B.
  • the lateral resolution of the scanning is the same as the beam width, which in this case is 20 microns.
  • the detection sensitivity of the in-line quadrature system can be measured by scanning over a single track multiple times and taking the difference between the scans. The detection sensitivity improves with averaging by the square root of the number of averages and can be as sensitive as 10 pm per laser spot before the averaging time takes too long and systematic drifts begin to dominate.
  • the detection sensitivity is 20 pm per laser spot with sixteen averages, which correspond to about 6 femtograms of minimum detectable protein mass per laser focus.
  • this mass sensitivity is scaled to 0.3 pg/mm 2 .
  • the protein pattern in Figure 19B is printed by a piezoelectric inkjet protein printer produced by Scienion Inc. and distributed by BioDot. Each spot is printed with 300 pL of protein solution, resulting in a 100 ⁇ m diameter spot on the isocyanate coating. Over 25,000 spots can be printed on a single silicon wafer, allowing room for highly multiplexed assays.
  • a disc was prepared with isocyanate coating and printed with more than 25,000 spots of mouse and rabbit IgG antigen, arranged in a radial pattern of grids, with 100 radial tracks along the radius and 256 spots in each track. The spots are grouped into 2x2 unit cells, in which two mouse spots are printed in one diagonal and two rabbit spots in the other diagonal.
  • the disc was first globally incubated in 10 ng/ml casein in a 10 mM Phosphate Buffered Saline (PBS) solution with 0.05% Tween 20, setting the baseline of the measurements.
  • PBS Phosphate Buffered Saline
  • the disc was then globally incubated with increasing concentrations from 100 pg/ml to 100 ng/ml of anti-mouse IgG, in PBS buffer with 0.05% Tween 20 and 10 ng/ml of casein. Each incubation lasted for 20 hours on a orbital shaker (VWR) to ensure the system reaches equilibrium and is not limited by mass transport to the disc surface.
  • VWR orbital shaker
  • the disc was scanned after each incubation.
  • the antibody- antigen binding was analyzed by first comparing each scan with the prescan before incubation, dividing the protein height changes by the prescan protein height for all the spots to get the ratio of height change for each spot, and then taking the difference of this height change ratio between the specific (mouse) and non-specific (rabbit) spots.
  • This relative difference in height change is defined as the assay signal.
  • an assay signal of 0.1 means that the specific spots gains 10% more mass than the nonspecific spots. This analysis provides good rejection of systematic shifts, wash-off effects, and non-specific binding that is common to both groups of spots.
  • FIG. 20A and 20B Histograms of assay signal in each unit cell as a function of dose are shown in Figure 2OA, and the dose response curve is shown in Figure 20B.
  • a dose response curve is obtained by fitting a Gaussian to each of the distributions, and the centers of the Gaussian fit are used as the average assay signals.
  • the error bars of the data points in Figure 20B are set by the standard error of the measurements.
  • the sensitivity of the current system is 100 pg/ml, when the dose response curve runs into the detection baseline. At this concentration level, the average detected protein mass change per spot is only 20 femto grams.
  • the dose response curve saturates at 16% mass increase, suggesting a 10-percent biological activity of the printed protein.
  • FIG. 21 shows the standard error of the assay versus the number of assays per disc. The error bars in this figure are set by the statistics over different wells of assays. The standard error increases as the square root of the number of assays, suggesting that the system is unbiased and that the measurement noise is uncorrelated.
  • the sensitivity limit of the assays can be obtained as a function of the number of assays per disc, as shown in Figure 22.
  • This detection limit has contributions from the noise increase and from the shape of the dose response curve. As an example, if thirty-two different assays were performed on this disc, then the detection limit for each assay would be 2 ng/ml. By further extrapolating this curve, if a single unit cell were treated as an independent assay, then the sensitivity of this assay would be about 10 ng/ml.
  • silicon dioxide grown thermally on silicon wafers was obtained with an oxide thickness of 80 nm, in condition for in-line detection. Proteins were spotted onto these wafers in individual spots using a Deerac printer. The far-field scanning in this case was ⁇ napeitured, collecting the full intensity. Clear modulation of the intensity is caused by the immobilized protein spots on the wafer surface as shown in Figures 23 and 24.
  • Figure 23 shows a cross-section across a single spot showing an outer ridge and internal ridges. The protein variation is resolvable down to 100 pm.
  • Figure 24 shows a high- resolution scan of a spot with a clear ring structure.
  • An alternative embodiment of the disk changes the oxide thickness from 80 nm to 120 nm.
  • the sign of the signal caused by molecular phase shifts contrasted to Rayleigh scattering (that removes light from the detected beam) are opposite.
  • the scattering of energy out of the reflected beam is negative, while added protein load on the surface is positive.
  • Scattering losses are always negative, while added protein load on a 120 nm oxide disk produces a positive shift in the intensity.
  • This principle has been demonstrated experimentally.
  • a scan of about 200 protein spots 120 micron diameter IgG spots on a 120 nm oxide biological disc
  • the protein spots (about 3 nm high) are bright, while the small dust and debris show as black specks.
  • Another feature of direct detection is reference subtraction which subtracts the common-mode effects such as non-specific binding.
  • the inline detection can use the principle of differential encoding, such as shown in U.S. Patent Application 11/345,566 entitled "Differentially Encoded Biological Analyzer Planar Array Apparatus and Methods," which was previously incorporated by reference.
  • One embodiment of differential encoding is the 2x2 unit cell shown in Figure 27.
  • the example of a "unit cell” shown in Figure 27 has target and reference spots placed in a 2x2 array.
  • the data on the right is data of unit cell spots of approximately 120 micron diameter printed onto a 120 nm oxide biological disc. Two similar proteins are spotted in a 2x2 array pattern.
  • One set is specific to the analyte, while the other set has similar properties, but is not specific to the analyte.
  • common non-specific binding increases both spot heights similarly, but the specific spot height increases more because of the specific binding to the analyte.
  • Figure 29 graphs sample data showing the detection sensitivity of in-line quadrature on a 120 nm oxide biological disc. The scan data on the upper left gives two line plots on the right, one through the center of an IgG spot, and the other on the so-called land. The roughness is converted into a mass sensitivity of about 0.27 pg/mm 2 .
  • the histogram in Figure 30 shows the root variance in the surface height between two scans of the same disc before and after a 20 hour buffer wash, which was determined to be 46 picometers per focal spot, corresponding to 5 femtograms of protein per focal spot with a diameter of 15-20 microns.
  • I mm which has the units of mass per length.
  • the scaling sensitivity is divided by the square-root of the sensing area. For a square millimeter this is:
  • This area-dependent sensitivity is comparable to the best values determined by surface plamon resonance (SPR). This sensitivity is gained without the need for resonance and hence is much more robust and easy to manufacture than other interferometric or resonance approaches.
  • the dose-response curve of a 120 nm oxide biological disc is obtained by printing spots in the 2x2 unit cell pattern on a disc.
  • An example of the spot layout is shown in Figure 31.
  • the disc is spotted with 25,600 spots in 100 radial steps and 256 angular steps. This produces 6,400 unit cells.
  • a dose-response curve was obtained by sequentially incubating the entire disc with increasing concentrations of analyte (anti-rabbit) in 10 ng/ml casein in PBS.
  • the resulting dose response curve is shown in Figure 32 using approximately 3,000 of the spots.
  • Figure 32 presents assay data showing change in spot mass as a function of analyte concentration for a series of incubations on a 120 nm oxide disc.
  • the smooth curve is a Langmuir function fit to the data.
  • the dynamic range between saturation and the limit-of-detection is about 300: 1.

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Abstract

L'invention concerne un procédé et un appareil destiné à être utilisé avec un faisceau sonde et un détecteur permettant de détecter la présence d'un analyte cible dans un échantillon. L'appareil compred un substrat et une biocouche située sur le substrat, destinée à réagir à l'analyte cible lorsque l'échantillon est déposé sur la biocouche. Le substrat peut être sélectionné de manière à réduire sensiblement la réflectance du substrat tout en conservant sensiblement la diffusion optique dans l'analyte cible. Le substrat peut être conçu de manière à ce que les ondes réfléchies par le substrat sont sensiblement en quadrature avec des ondes diffusées par l'analyte cible; ou de manière à ce que les ondes réfléchies par le substrat et diffusées par l'analyte cible interfèrent dans le champ éloigné et créent directement une modulation d'intensité détectable par le détecteur. La biocouche peut comprendre une pluralité de points et les points peuvent être groupés en cellules d'unités présentant des anticorps spécifiques et des anticorps non spécifiques pour la mise en réaction de l'analyte cible.
EP07757051A 2006-02-16 2007-02-15 Quadrature en ligne et detection interferrometrique en quadrature de phase amelioree antireflet Withdrawn EP1987347A1 (fr)

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